ABSTRACT Staphylococcus aureus colonizes the nasopharynx and gastrointestinal tract of humans, which represents a key risk factor for invasive diseases such as skin and bloodstream infections. Hallmarks of S. aureus are the persistent colonization of about one third of the human population and the ability to cause reiterative infections without eliciting protective immune responses. Earlier work reported that the type VII secretion system (ESS or T7bSS) is crucial for the pathogenesis of S. aureus bloodstream infections and staphylococcal escape from T cell responses. During infection, S. aureus activate dendritic cells to secrete IL-12 (p40/p70), thereby compromising the induction of adaptive immune responses due to IL-12 skewing of g- interferon-producing TH1 cells, which impede TH2 polarization and the host's ability to produce pathogen-specific antibodies. Earlier work identified genes (esaA, essA, essB and essC), whose products function as core components of the T7bSS in the staphylococcal membrane, which associate with mobile proteins (EsxA, EsxB, EsxC, EsxD and EssD) that travel the ESS pathway. One of these secretion substrates, EssD comprises a nuclease domain that cleaves DNA and promotes IL-12 secretion in myeloid cells in vivo and in vitro. We also identified new genes whose products are essential for ESS secretion. The product of essH is secreted via the Sec pathway and exhibits peptidoglycan hydrolase activity required for the assembly of the ESS pathway. Genes whose products are members of the DUF5079 and DUF5080 family represent membrane proteins that contribute to T7bSS pathway. Our proposal seeks to investigate the trafficking of the EssH effector into myeloid cells, to locate its nuclease activity in subcellular compartments of myeloid cells and to identify host signaling components required for IL-12 secretion. Other work will examine the mechanistic contributions of EssH, DUF5079 and DUF5080 proteins towards assembly and secretion of the T7bSS. Last, we will study S. aureus WU1, an ST88 isolate that adapted to persistently colonize the nasopharynx and gastrointestinal tract of mice and to cause spontaneous invasive diseases. Our experiments will explore the contributions of the ESS pathway during S. aureus WU1 persistent colonization and invasive disease in mice.